1. Field of the Invention
This invention relates to novel recombinant nucleic acids encoding the enzyme dihydrofolate reductase (DHFR) from mycobacteria, to novel recombinant DHFR peptides produced by such sequences, and to vaccines, diagnostic kits, cells and therapies utilizing these peptides and nucleic acid sequences. The invention is also directed to methods for using the sequences and peptides to develop drugs specific to M. avium and other species of mycobacteria, to identifying other DHFR sequences and peptides, as well as diagnostic and treatment methods incorporating the disclosed sequences and peptides.
2. Description of Background
The Mycobacterium avium complex represents one of the most serious opportunistic infections and is often associated with advanced stages of autoimmune deficiency syndrome or AIDS (J. J. Ellner et al., J. Infect. Dis. 163:1326-35, 1991; J. A. Havlok, Jr. et al., J. Infec. Dis. 165:577-80, 1992; C. C. Hawkins et al., Ann. Intern. Med. 105:184-88, 1986; D. S. O""Brien et al., Am. Rev. Respir. Dis. 135:1007-14, 1989; N. Rastogi et al., Res. Microbiol. 145:167-261, 1994). Unlike Mycobacterium tuberculosis, which can be successfully treated with two or three drug combinations (except for multidrug resistant M. tuberculosis; MDR-TB), the M. avium complex is resistant to many antimycobacterial agents (B. D. Agins et al., J. Infect. Dis. 159:784-87, 1989; C. Benson et al., Sixth International Conference on AIDS, San Francisco, 1990; J. Chiu et al., Ann. Intern. Med. 113:358-61, 1990; F. De Lalla et al., Antimicrob. Agents Chemother. 36:1567-69, 1992; L. Heifets et al., Antimicrob. Agents Chemother. 37:2364-70, 1993; D. Y. Rosenzweig, Amer. Rev. Resp. Dis. 113(Suppl.):55, 1976). Drug resistance in M. avium is still considered an inherent property of the wild type organism (S. L. Morris et al., Complex. Res. Microbiol. 147:68-73, 1996), resulting in large part from the refractory nature of the organism""s cell envelope (H. L. David, Rev. Infect. Dis. 3:878-84, 1981; N. Rastogi et al., Res. Microbiol. 145:243-52, 1994; N. Rastogi et al., Antimicrob. Agents Chemother. 20:666-77, 1981). Although M. aviuminfections in AIDS patients are treated with 3-6 different drugs, the long term prognosis is still poor (B. D. Agins et al., J. Infect. Dis. 159:784-87, 1989; C. Benson et al., Sixth International Conference on AIDS, San Francisco, 1990; J. Chiu et al., Ann. Intem. Med. 113:358-61, 1990; F. De Lalla et al., Antimicrob. Agents Chemother. 36:1567-69, 1992; J. J. Ellner et al., J. Infect. Dis. 163:1326-35, 1991).
Tuberculosis is a disease of worldwide significance and notoriety. At any one time, about one-third of the world is infected with M. tuberculosis resulting in eight million new cases of tuberculosis and 2.9 million deaths annually (A. Arachi, Tubercle. 72:1-6, 1991). It is estimated that about 0.3% of U.S. residents are infected and at risk to develop active disease (CDC 1996, CDC Revises HIV Infection Estimates. HIV/AIDS Prevention. August:2). This risk becomes even greater if the person is co-infected with the human immunodeficiency virus (HIV). If so, estimates indicate that progression to tuberculosis will occur in about 30% of those cases and the risk for developing tuberculosis becomes 113 times greater (tuberculosis., N.a.p.t.c.m.-r., MMWR. 41 RR-11:1-71, 1992).
As the projected figure for HIV infections is more than 20 million by the year 2000, it is probable that the number of tuberculosis cases worldwide will also increase. Even in 1991, the figure for people co-infected with HIV and M. tuberculosis was estimated to be 3.1 million (J. F. Murray, Bull. Int. Union Tuberc. Lung Dis. 66:21-15, 1991). In addition, life-threatening strains of MDR-TB are appearing. Some of these strains can result in a high mortality rate (e.g. 72-89%), with death occurring in a short period (e.g. 4-16 weeks) (CDC, Mortal. Morbid. Weekly Rep. 39:718-22, 1990; CDC, Mortal. Morbid. Weekly Rep. 40:649-652, 1991; B. R. Edlin et al., New Engl. J. Med. 326:1514-21, 1992). In summary, the impact of tuberculosis on the world today can best be appreciated by the fact that the World Health Organization declared tuberculosis a global public health emergency, a distinction never before given to any other disease (WHO., Soz Praventivmed. 38:251-52, 1993). Consequently, the development of new antimycobacterial drugs is an important research endeavor.
The dihydrofolate reductase (DHFR) enzyme is an important target for medicinal chemistry (K. Bowden et al., J. Chemother. 5:377-88, 1993) and DHFR inhibitors have been used in anticancer therapy (e.g. methotrexate (W. A. Bleyer, Cancer Treat. Rev. 41:36-51, 1978)), antibacterial therapy (e.g. trimethoprim (M. Finland et al., J. Infect. Dis. 128:S425-816, 1973)), and antimalarial therapy (e.g. pyrimethamine (A. K. Saxena, Prog. Drug Res. 30:221-80, 1986)). Dihydrofolate reductase is present in all cells and is necessary for the maintenance of intracellular folate pools in a biochemically active reduced state (M. McCourt et al., J. Am. Chem. Soc. 113:6634-39, 1991). Inhibition of the enzyme is effective because binding affinities for substrate analogs are so great that such analogs are not readily displaced by the natural substrates. Enzyme inhibition results in the depletion of intracellular reduced folates that are required for one carbon transfer reactions, which in turn are important for the biosynthesis of thymidylate, purine nucleotides, methionine, serine, glycine and many other compounds needed for RNA, DNA, and protein synthesis. FIG. 1 depicts DHFR""s role in the biosynthesis of tetrahydrofolate and cell metabolism. (P. G. Hartman, J. Chemother. 5:369-76, 1993). Some bacteria have an uptake system for folates, but most have to synthesize folates de novo by reduction of dihydrofolate to tetrahydrofolates.
Although DHFR is not a new drug target, enthusiasm in the development of improved derivatives to inhibit DHFR is very intense, (D. P. Baccanari et al., J. Chemother. 5:393-99, 1993; K. Bowden et al., J. Chemother. 5:377-88, 1993; M. McCourt et al., J. Am. Chem. Soc. 113:6634-39, 1991; J. R. Piper et al., J. Med. Chem. 39:1271-80, 1996; B. I. Schweitzer et al., FASEB. 4:2441-52, 1990; J. K. Seydel, J. Chemother. 5:422-29, 1993), and particularly with regard to mycobacteria (K. H. Czaplinski et al., Eur. J. Med. Chem. 30:779-87, 1995; M. Kansy et al., Eur. J. Med. Chem. 27:237-44, 1992; H. H. Locher et al., Antimicrob. Agents Chemother. 40:1376-81, 1996; S. C. C. Meyer et al., Antjiicro. Agents Chemother. 39:1862-63, 1995; R. L. Then, J. Chemother. 5:361-68, 1993). A unique feature of DHFR is the selectivity possible in the design of inhibitors for this target, thus making it an ideal target for antimycobacterial agents using rational and effective drug design. Although genes for DHFR (fol A) have been identified in other bacteria, they are not equivalent to the fol A gene from M. avium or other mycobacteria. The enzyme product of the M. avium gene (i.e. DHFR) is structurally different from other known DHFRs. For these reasons and others having to do with, for example, toxicity, selective drugs can be designed for individual species. Thus, a selective drug for M. avium can be designed in such a way that it will not affect DHFRs in other species, such as humans. One example of the species specific nature of the enzyme is demonstrated by the fact that the diaminopyrinidines, when properly substituted, can be several thousand times more active against bacterial than mammalian DHFR (P. G. Hartran, J. Chemother. 5:369-76, 1993). In addition, there are many possible inhibitors of this enzyme that have not been synthesized or studied (K. Bowden et al., J. Chemother. 5:377-88, 1993). DHFR also represents an enzyme that has been extensively used in the development of site directed inhibitors based upon X-ray crystallographic and molecular graphic studies (K. Bowden et al., J. Chemother. 5:377-388, 1993; M. P. Bradley, J. Med. Chem. 36:3171-77, 1993; B. J. Denny et al., J. Med. Chem. 35:2315-20, 1992; M. McCourt et al., J. Am. Chem. Soc. 113:6634-39, 1991; B. Roth, FASEB. 45:2765-72, 1986; W. M. Southerland, J. Computer-Aided Molecular Design. 8:113-22, 1994).
An important objective in the future development of DHFR inhibitors will be improving delivery of antimycobacterial drugs. One research program, designed to develop sustained and targeted delivery of first-line antituberculosis drugs using micro-encapsulation techniques has successfully formulated a micro-encapsulated form of rifampicin that shows good release characteristics (W. W. Barrow et al., European Society for Mycobacteriology, Institute Pasteur, Paris, France: 1996:50). Use of this formulation has resulted in reduction in colony forming units (CFUs) in both the M. tuberculosis H37Rv infected macrophage and mouse models, suggesting that this technology can also be used for other antimycobacterial drugs including the lipophilic DHFR inhibitors.
The history of the development of antifolates is long and includes significant contributions to that area of anticancer and anti-infective drug discovery. Improved agents against opportunistic infections have recently been developed including a synthetic process to prepare 5-alkyl-5-deaza analogs of antifolates, both classical and the lipophilic types. These analogs have been used in several studies (J. R. Piper et al., J. Med. Chem. 39:1271-80, 1996).
Mycobacterial DHFR has been a target for drug design for about three decades. Two groups in particular have synthesized lipophilic antifolates targeting this enzyme in mycobacteria. One group published activity results on a small number of 2,4-diamino-6-substituted pteridines, 6-substituted 8-deazapteridine, 5,6-substituted-5-deazapteridines and 5-methyl-6-substituted-5,8-di-deazapteridines (quinazolines) against M. species 607 (W. T. Colwell et al., Chemistry and Biology of Pteridines. vol. Elsevier North Holland, Inc., Amsterdam. 215-18, 1979; J. I. DeGraw et al., J. Medicinal Chem. 17:144-46, 1974; J. I. DeGraw et al., J. Medicinal Chem. 17:762-64, 1974). Another group published several papers describing the design and synthesis of substituted 2,4-dianino-5-benzyl pyrimidines (Trimethoprim analogs) active against mycobacterial DHFR from M. lufu and screened in vitro against M. lufu, M. tuberculosis and M. marinum (K. H. Czaplinski et al., Eur. J. Med. Chem. 30:779-87, 1995; M. Kansy et al., Eur. J. Med. Chem. 27:237-44, 1992; J. K. Seydel et al., Chemother. 29:249-61, 1983).
These data support the activity of folate analogs against mycobacteria. Active compounds were obtained in each series of experiments. The quinazoline analogs were targeted as lead compounds. Although quite active against isolated DHFR, these compounds demonstrated poor selectivity. However, it was apparent that selectivity was enhanced by a 5-methyl substitution for both the 5-deaza- and 5,8-di-deazapteridines (quinazolines). Although low toxicity was noted for the quinazolines in mice, equivocal results against M. leprae in the mouse foot pad model were noted (W. T. Colwell et al., Chemistry and Biology of Pteridines, Elsevier North Holland, Inc., Amsterdam. 215-18, 1979).
One research group has been pursuing trimethoprin analogs such as 2,4-diamino-5-benzylpyrimidine derivatives (M. Kansy et al., Eur. J. Med. Chem. 27:237-44, 1992; J. K. Seydel, J. Chemother. 5:422-29, 1993; J. K. Seydel et al., Chemother. 29:249-61, 1983). Several 4xe2x80x2-modified derivatives have been synthesized to extend into the glutamate binding region of the enzyme (L. F. Kuyper et al., J. Med. Chem. 28:303-11, 1985). A small number of these compounds showed very good selective activity against M. lufu in vitro and against the isolated bacterial DHFR (K. H. Czaplinski, Eur. J. Med. Chem. 30:779-87, 1995). The compounds showed an activity profile against M. tuberculosis in vitro (M. Kansy et al., Eur. J. Med. Chem. 27:237-44, 1992).
The efficacy of using antifolates against M. avium and other mycobacteria has not yet been fully determined. In addition, as new antifolates are developed, their efficacy against M. avium and other mycobacteria will need to be evaluated. Such studies could be greatly facilitated through the use of purified recombinant mycobacterial DHFR.
Thus far, there has not been complete or accurate identification, sequencing and cloning of the mycobacterial DHFR gene of any species. Two papers have been published about mycobacterial DHFR. Al-Rubeai et al. (M. Al-Rubeai et al., Biochem. J. 235:301-3, 1986), reported on the purification and characterization of DHFR from M. phlei and Sirawarapom et al. (W. Sirawaraporn et al., Exper. Parisitol. 72:184-90, 1991), reported on the purification and characterization of DHFR from a strain of M. smegmatis. The reported molecular weights were 15 and 23 kDa for M. phlei, and M. smegmatis DHFR, respectively. In Sirawaraporn et al., the authors reported on the amino terminal sequencing of the protein. Of the fifteen assignments reported, twelve were stated to be clear, one ambiguous and two could not be determined. Other than this incomplete and partial sequencing of M. segmatis DHFR protein, no mycobacterial DHFR DNA sequences have previously been reported.
The invention is directed to all or portions of novel recombinant nucleic acids encoding, in whole or in part, a dihydrofolate reductase (DHFR) peptide from mycobacteria such as M. avium or M. tuberculosis, to novel recombinant DHFR peptides produced by such sequences, and to vaccines, diagnostic kits, cells and therapies utilizing these peptides and nucleic acid sequences. The invention is also directed to methods for using the sequences to develop drugs specific to M. avium and other mycobacteria, to identify and sequence corresponding sequences in other species of Mycobacterium, as well as to diagnostic and treatment methods incorporating the disclosed sequences and peptides.
One embodiment of the invention is directed to recombinant nucleic acids comprising all or a portion of the nucleic acid sequence that encodes a mycobacterial DHFR protein, such as the DHFR protein of Mycobacterium avium, Mycobacterium bovis, Mycobacterium tuberculosis or Mycobacterium leprae. The nucleic acid may comprise DNA, RNA or PNA, and may include additional sequences to direct transcription or translation, such as a promoter, a polymerase binding site, an enhancer, or a transcription or translation termination site. The nucleic acid may encode portions of the DHFR protein, such as an enzymatically active portion or antigenically active portion. Alternatively, the sequence may encode the entire amino acid sequence of the DHFR protein.
Another embodiment of the invention is directed to vectors comprising one of these recombinant nucleic acids, or a recombinant cell containing one of these nucleic acids. The nucleic acid may be integrated into the cell""s genome, or it may be episomal. The cell may be prokaryotic or eukaryotic.
Another embodiment of the invention is directed to recombinant peptides comprising an amino acid sequence containing all or portion of a mycobacterial DHFR protein, such as M. avium, M. bovis, M. tuberculosis or M. leprae protein. The recombinant peptide may encode only an enzymatically or antigenically active portion of the peptide, or the entire amino acid sequence of the protein.
Another embodiment of the invention is directed to methods for screening for an agent which inhibits the activity of recombinant DHFR. This method comprises determniing the activity of DHFR protein upon incubation with a plurality of agents and selecting the agent that inhibits the activity. The DHFR protein may be derived from M. avium, M. bovis, M. leprae, M. tuberculosis or other mycobacteria. The DHFR protein may be an entire DHFR protein or comprise only selected enzymatically active or antigenically active portions thereof. The plurality of agents may be, for example, over 102 different agents, and may be drawn from a collection of related chemical compounds, including chemical modifications of folate, methotrexate, trimethoprin or combinations thereof. The incubation may comprise mixing the DHFR protein with the agents under conditions allowing for molecular interaction, such as binding, inhibition of enzymatic activity or inhibition of immunogenicity. A preferred embodiment of this method includes the steps of selecting a plurality of agents that inhibit the Mycobacterium DHFR protein, determining the molecular conformation of each agent, and identifying a common inhibitory molecular conformation.
Another embodiment of the invention relates to methods for assessing the ability of an agent to inhibit the activity of a DHFR protein comprising the steps of incubating a recombinant mycobacterial DHFR protein with the agent, determining the activity of the incubated DHFR protein, and comparing the activity with the wild-type activity of the protein. In this method, the activity may be enzymatic activity or immunogenic activity. The agent may be incubated by simply mixing the agent with the protein under, for example, physiological conditions. The agent preferably is usefuil for treatment of a mycobacterial infection. The DHFR protein may be a protein produced by a species selected from the group of M. avium, M. bovis, M. tuberculosis, M. leprae, or another mycobacteria, such that the agent is specific for treatment of one of these same species.
Another embodiment of the invention is directed to methods for selecting an antimycobacterial agent specific against a mycobacterial infection comprising crystallizing a recombinant Mycobacterium DHFR protein and determining the molecular conformation of the protein, identifying a binding site within the molecular conformation, and selecting the agent with the molecular structure that fits the binding site. The binding site is preferably a substrate binding site.
Another embodiment of the invention is directed to methods for identifying the sequence of a mycobacterial DHFR gene comprising amplifying nucleic acid in a biological sample containing Mycobacterium by a polymerase chain reaction with two probes which span all or part of the M. avium DHFR gene, and determining the sequence of the amplified nucleic acid to identify the sequence of the mycobacterial DHFR gene. Preferably, the sequence identified encodes the DHFR protein of M. avium, M. bovis, M. tuberculosis or M. leprae. In a preferred embodiment, the sequence of the first probe contains a sequence from the 5xe2x80x2 terminus of the M. avium gene and the sequence of the second probe contains a sequence from the 3xe2x80x2 terminus of the M. avium gene.
Another embodiment is directed to methods for detecting Mycobacterium infection such as infection with M. avium, M. bovis, M. tuberculosis or M. leprae, by contacting a biological sample obtained from a patient with an antibody specific to a recombinant Mycobacterium protein, and detecting bound antibody in the sample. The biological sample may be a sample of bodily fluid from a human. The antibody may be a monoclonal or polyclonal antibody.
Another embodiment of the invention is directed to methods for detecting a Mycobacterium infection by contacting a biological sample obtained from a patient with a recombinant mycobacterial protein or an active portion thereof, and detecting protein bound with antibody from the patient in the sample. The infection may be an infection of M. avium, M. bovis, M. tuberculosis or M. leprae. In this embodiment, the protein may be labeled with a detectable label, such as a radioisotope, stable isotope, fluorescent chemical moiety, enzyme, metal or combinations thereof.
Another embodiment of the invention is directed to methods for detecting a mycobacterial infection comprising amplifying nucleic acid in a biological sample containing Mycobacterium by a polymerase chain reaction comprising two probes, wherein the two probes span all or part of the M. avium DHFR gene, and detecting amplified nucleic acid that corresponds to an amplification of the nucleic acid between the two probes. In a preferred embodiment, the sequence of the first probe contains a sequence from a terminus of the M. avium gene and the sequence of the second probe contains a sequence from the opposite terminus of the M. avium gene. The two probes may be labeled with detectable labels, such as radioisotopes, stable isotopes, fluorescent chemical moieties enzymes, metals and combinations thereof. In one embodiment, the step of detecting comprises determining the size of the nucleic acid amplified.
Another embodiment of the invention is directed to methods for screening for an agent that interacts with M. avium DHFR protein by immunizing an animal with a protein containing at least a portion of the M. avium DHFR protein to generate anti-protein antibodies, immunizing another animal with the anti-protein antibodies to generate a collection of anti-idiotypic antibodies, selecting a anti-idiotypic antibody of the collection that binds to dihydrofolate, and identifying an agent that binds to said anti-idiotypic antibody. In this embodiment, the portion of the protein may be an enzymatically active or antigenically active portion, or a conserved region of the DHFR protein. The anti-idiotypic antibody may have an affinity for dihydrofolate that is comparable to the affinity of the catalytic site of M. avium DHFR for dihydrofolate. Preferably, the portion is a peptide corresponding to a region of mycobacterial DHFR protein that is not present in mammalian DHFR protein.
Another embodiment of the invention is directed to methods for detecting M. avium in a sample comprising immunizing an animal with a protein containing the DHFR sequence of the present invention to generate antibodies specific to the sequence, immunizing another animal with the antibodies to generate anti-idiotypic antibodies, and detecting M. avium DHFR protein in an immunoassay containing said anti-idiotypic antibodies. In this embodiment, the immunoassay may be a competitive immunoassay, an indirect immunofluorescence assay, an ELISA assay, an immunoprecipitation assay or other well-know or useful assay.
Another embodiment of the invention is directed to methods of detecting M. avium DHFR in a biological sample comprising the steps of combining a portion of the sample with an idiotypic antibody to M. avium DHFR protein, an anti-idiotypic monoclonal antibody to the idiotypic antibody such that the anti-idiotypic monoclonal antibody exhibits structural congruence with at least one epitope of the protein to form an assay mixture in which there is competition between the protein and the anti-idiotypic monoclonal antibody for binding to the anti-idiotypic antibody, and detecting M. aviumDHFR protein in the sample by determining the amount of bound labeled antibodies disposed within the anti-idiotypic antibody pairs. In one embodiment, determination of the amount of bound labeled antibody disposed within the anti-idiotypic antibody pairs follows a separation of the anti-idiotypic pairs from unbound antibody. Such separation may be achieved by precipitation. In one embodiment, at least one component of the mixture may be labeled with a detectable label, such as a fluorophore, radioactive compound, chemiluminescent compound, latex bead, enzyme, enzyme cofactor or enzyme inhibitor. The idiotypic antibody may be attached to a substrate or, alternatively, the anti-idiotypic antibody may be attached to a substrate.
Another embodiment of the invention is directed to antibodies specifically reactive against DHFR peptides of the invention. Antibodies may be polyclonal or monoclonal and expressed from a population of hybridoma cells. Such antibodies may be reactive against specific epitopes of the peptide such as the substrate binding site.
Another embodiment of the invention is directed to vaccines and diagnostic kits incorporating recombinant Mycobacterium DHFR peptides or antibodies specifically reactive against these recombinant peptides and to methods for using such vaccines and diagnostic kits.
Other embodiments and advantages of the invention are set forth, in part, in the description which follows, and, in part, will be obvious from this description and may be learned from the practice of the invention.